gender: female strain: Sprague Dawley developmental stage: adult tissue: prefrontal cortex prenatal diet: control adult treatment: adjuvant injection termination time point (post injection): day 16 animal id: 75 sample type: sample
Treatment protocol
Breeding and prenatal alcohol exposure: Rats were obtained from the Animal Care Center at the University of British Columbia, and were group- housed 1-2 weeks before breeding, with ad libitum access to standard lab chow (Jamieson’s Pet Food Distributors, Ltd., Delta, BC, Canada). Details of the procedures for breeding and handling have been published previously (Glavas et al. 2007). Briefly, females and males were co-housed in stainless steel cages with mesh front and floors, and wax paper under the cages was checked daily for the presence of vaginal plugs, which indicated day 1 of gestation. Thereafter pregnant dams were singly housed, and were assigned to one of three groups – a control group (C; fed laboratory chow ad libitum), pair-fed group (PF; liquid-control diet, with maltose-dextrin isocalorically substituted for ethanol, in the amount consumed by an ethanol- consuming partner, matched for g/kg body weight/day of gestation), or an ethanol-fed group (E; ad libitum access to liquid ethanol diet, with 36% calories derived from ethanol). All animals had ad libitum access to water, and diets were fed from gestation days 1-21 (Weinberg/Kiever Ethanol Diet #710324, Weinberg/Kiever Control Diet #710109, Dyets Inc., Bethlehem, PA). After gestational day 21, all animals were given laboratory chow and water ad libitum. Litters were weighed and culled at birth to 5 males and 5 females, when possible. Following weaning (on postnatal day 22) female offspring were group-housed by litter (2-3 rats per cage) until the start of testing. Induction of adjuvant-induced arthritis: Details of the postnatal induction of adjuvant-induced arthritis in these animals have been previously published (Zhang et al. 2012). Briefly, female offspring (50-65 days of age) from the C, PF, and E groups were divided into two postnatal treatment groups, in which animals received an intradermal injection at the base of the tail of either 0.1 ml of 12 mg/ml suspension of complete Freund’s adjuvant (CFA) (Adjuvant group), or 0.1ml saline (Saline group). All animals were single-housed after injection, and monitored for clinical signs of arthritis, from the onset of AA through to resolution. To evaluate clinical signs of arthritis, animals were lightly anesthetized with isofluorane, and paws were scored for severity of redness and swelling, on days 7, 10, and every other day afterwards, following injection (results published previously in Zhang et al., 2012). Termination of animals: Animals were terminated in two cohorts for analysis of gene expression. One cohort was terminated on day 16 post-injection (at the peak of adjuvant-induced arthritis), and another on day 39 post-injection (during resolution phase of arthritis). Each cohort contained 27 adjuvant-injected animals (9 each of C, PF, and E) and 15-18 saline-injected animals (5-6 each of C, PF, and E). For termination, animals were singly removed from their colony room, exposed to CO2 for 30 seconds, and then quickly decapitated. Brains were rapidly removed, immediately frozen on dry ice, wrapped in foil, and stored at -70 °C.
Extracted molecule
total RNA
Extraction protocol
Brains were gradually thawed to 4 °C, and the prefrontal cortex (PFC) and hippocampus (HPC) were dissected using RNase-free technique. Dissected tissues were placed in RNAlater and stored at -20 °C. Total RNA and DNA were simultaneously extracted from the dissected tissues using the Qiagen AllPrep DNA/RNA Mini kit, and a DNase digestion step was included in the RNA extraction process. RNA integrity was determined using the Agilent BioAnalyzer mRNA Nano assay, and no samples were excluded due to low RNA integrity. Only one sample was excluded at this stage (PF hippocampus from the Day 39 Adjuvant group), due to suspected contamination.
Label
Cy3-SA
Label protocol
To generate cRNA for microarray analysis, 250 ng of total RNA from each sample was amplified using the Ambion Illumina TotalPrep RNA Amplification kit, in batches of ~24 samples at a time. Samples were distributed across amplification batches such that batch was not confounded with experimental treatment group.
Hybridization protocol
Gene expression was analyzed for both the PFC and HPC using the Illumina RatRef-12 Expression BeadChip microarray, which has 12 arrays per chip. PFC and HPC samples were run separately on different dates, due a limit of processing 8 chips (96 arrays) per batch. 750 ng of cRNA was applied to each array, with one sample per array. Experimental groups were counter-balanced across the arrays, such that chip batch was not confounded with any experimental treatment group, amplification batch, or dissection batch. Additional arrays included an amplification replicate and several hybridization replicates, yielding a total of 96 arrays run per tissue. Microarrays were scanned on the Illumina iScan, and bead-level expression data was collected.
Scan protocol
Standard Illumina scanning protocol
Description
Sample name: 5398636039_K
Data processing
Detection p-values were identified using calculateDetection in the R package beadArray. Data were then filtered to remove non-gene probes and processed by quantile normalization using the normalizeIllumina function of the beadArray package in R.
Submission date
Nov 21, 2014
Last update date
Nov 21, 2014
Contact name
Michael S. Kobor
Organization name
Centre for Molecular Medicine and Therapeutics / University of British Columbia
